Factor IX, a plasma protein involved in blood coagulation, is defective in patients with hemophilia B. This application examines the g- carboxyglutamic acid-rich domain of Factor IX to understand its role in membrane binding and in complex formation with Factor VIIIa. Two themes are the focus of this application: the nature of Factor IX- membrane interaction in chemical terms and of the Factor IXa-Factor VIIIa complex on membrane studies. We will explore the membrane binding properties of Factor IX, particularly the contribution of phosphatidylethanolamine and soluble phospholipids. Based on our X-ray crystal structure of pro-thrombin bound to phosphatidylserine, we will examine the homologues residues in Factor IX that form contact sites with phospholipid by site-specific mutagenesis. P-Benzoylphenylalanine-FIX (1-47) will be used as photoaffinity labels of platelet membranes to identify the protein or lipids to which Factor IXa binds. Based on our successful application of 2D NMR spectroscopy to the determination of the soluble structure of the Gla domain of Factor IX, we will determine the solution structure of the ternary Factor IX (1-47) phospholipid: calcium complex. The focus will be on defining the membrane finding site in Factor IX and the contact residues that determine specificity for acidic phospholipids. The metal generate Factor IX crystals in the presence of calcium phosphatidylserine suitable for X-ray crystallography. The solution structure of the Gla domains of other vitamin K-dependent proteins will be determined in the presence of phosphatidylserine in order to compare their interaction with phospholipid to that of Factor IX. Finally, Factor IXa-Factor VIIIA complex formation will be studied to understand formation of the Xase complex on membrane surfaces. Based upon the preliminary observation that a membrane finding peptide based upon Factor VIII greatly accelerates Factor X activation by Factor IXa in the absence of Factor VIIIa and phospholipid, we will determine the sites on Factor IXa and Factor X to which this peptide binds using a p-Bpa analog of FVIII (2303-2323) as photoaffinity label. The interaction of FVIII (2303-2323) with Factor IXa and Factor X as well as with FIX (1-47) will be characterized by UV difference and NMR spectroscopy. A high affinity peptide inhibitor of Factor IXa-Factor VIIIa interaction will be identified by screening a combinatorial phase library. Inhibitory peptides will be characterized for their mechanism of inhibition of binding of Factor IXa with Factor VIIIa. Understanding the details of Factor IX-membrane interaction and Factor IXa-Factor VIIIa complex formation will contribute to our knowledge of hemophilia and the treatment of thrombotic disease.